Oligonucleotide conjugates

ABSTRACT

Oligonucleotide conjugates, where an oligonucleotide is covalently attached to an aromatic system, are provided. In particular embodiments the oligonucleotide is complementary to the RNA component of human telomerase and is covalently attached to a nucleobase via an optional linker. The conjugates inhibit telomerase enzyme activity.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT/US02/09138, filed Mar.21, 2002, designating the U.S. and to be published in English 18 monthsafter the first priority date pursuant to Article 21(2) of the PatentCooperation Treaty. This application also claims priority to U.S.provisional patent application No. 60/278,322, filed Mar. 23, 2001. Bothof these priority applications are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to covalent oligonucleotide conjugates.More particularly, the present invention is directed to covalentoligonucleotide conjugates that are designed to inhibit the activity oftelomerase, an enzyme that is preferentially expressed in, and requiredfor the growth of, cancer cells.

BACKGROUND OF THE INVENTION Oligonucleotide Chemistry

Nucleic acid polymer chemistry has played a crucial role in manydeveloping technologies in the pharmaceutical, diagnostic, andanalytical fields, and more particularly in the subfields of antisenseand antigene therapeutics, combinatorial chemistry, branched DNA signalamplification, and array-based DNA diagnostics and analysis. Much ofthis chemistry has been directed to improving the binding strength,specificity, and nuclease resistance of natural nucleic acid polymers,such as DNA. Unfortunately, improvements in one property, such asnuclease resistance, often involve trade-offs against other properties,such as binding strength. Examples of such trade-offs abound: peptidenucleic acids (PNAs) display good nuclease resistance and bindingstrength, but have reduced cellular uptake in test cultures (e.g. Hanveyet al., Science, 258:1481-1485, 1992); phosphorothioates display goodnuclease resistance and solubility, but are typically synthesized asP-chiral mixtures and display several sequence-non-specific biologicaleffects (e.g. Stein et al., Science, 261:1004-1012, 1993);methylphosphonates display good nuclease resistance and cellular uptake,but are also typically synthesized as P-chiral mixtures and have reducedduplex stability, and so on.

Recently, a new class of oligonucleotide analog has been developedhaving so-called N3′→P5′ phosphoramidate internucleoside linkages, whichdisplay favorable nucleic acid binding properties, nuclease resistance,and water solubility (Gryaznov and Letsinger, Nucleic Acids Research,20:3403-3409, 1992; Chen et al., Nucleic Acids Research, 23:2661-2668,1995; Gryaznov et al., Proc. Natl. Acad. Sci., 92:5798-5802, 1995; andGryaznov et al., J. Am. Chem. Soc., 116:3143-3144, 1994). Uniformlymodified phosphoramidate compounds contain a 3′-amino group at each ofthe 2′-deoxyfuranose nucleoside residues replacing a 3′-oxygen atom. Thesynthesis and properties of oligonucleotide N3′→P5′ phosphoramidates arealso described in U.S. Pat. Nos. 5,591,607; 5,599,922; 5,726,297; and5,824,793.

Oligonucleotides conjugated to a signal generating system have been usedas tools in diagnostic applications, such as fluorescent in situhybridization (FISH). For example, specific microorganism (U.S. Pat. No.5,776,694) or telomerase-expressing cells (U.S. Pat. No. 5,891,639) wereidentified by labeling nucleic acids that were complementary tosequences unique to the target organism or the telomerase enzyme,respectively, and then contacting the probe with the target. Once theprobe had formed a hybrid with the target, the hybrid was detected byactivating the signal generating system that was bound to the probe. Inanother use, labeled probes were used in DNA microarray experiments (SeeU.S. Pat. No. 6,040,138). Typically, probes from a biological samplewere amplified in the presence of nucleotides that had been coupled to areporter group, such as a fluorescent label, thereby creating labeledprobes. The labeled probes were then incubated with the microarrays sothat the probe sequences hybridized to the complementary sequencesimmobilized on the microarray. A scanner was then used to determine thelevels and patterns of fluorescence.

The present invention relates to a new class of oligonucleotideconjugates having telomerase inhibiting activity.

Telomerase

Telomerase is a ribonucleoprotein that catalyzes the addition oftelomeric repeat sequences to chromosome ends. See Blackburn, 1992,Annu. Rev. Biochem., 61:113-129. There is an extensive body ofliterature describing the connection between telomeres, telomerase,cellular senescence and cancer (for a general review, see Oncogene,volume 21, January 2002, which is an issue focused on telomerase).Telomerase has therefore been identified as an excellent target forcancer therapeutic agents (see Lichsteiner et al., Annals New York Acad.Sci. 886:1-11, 1999)

Genes encoding both the protein and RNA components of human telomerasehave been cloned and sequenced (See U.S. Pat. Nos. 6,261,836 and5,583,016, respectively) and much effort has been spent in the searchfor telomerase inhibitors. Telomerase inhibitors identified to dateinclude small molecule compounds and oligonucleotides. By way ofexample, WO01/18015 describes the use of oligonucleotides that compriseN3′→P5′ thio-phosphoramidate internucleoside linkages, and which arecomplementary to the sequence of the human telomerase RNA component, toinhibit telomerase activity.

SUMMARY OF THE INVENTION

The compositions and methods of the present invention relate tocompounds comprising an oligonucleotide and at least one covalentlylinked group, preferably an aromatic system. The sequence of theoligonucleotide is selected to be complementary to the RNA component oftelomerase, and the covalently linked aromatic system can be, forexample, a polyaromatic hydrocarbon, a mono- or poly-cyclicheteroaromatic system, or a nucleobase (including modified nucleobasesand nucleobase analogs). The aromatic group is typically, although notnecessarily, covalently attached to either the 3′-position or the5′-position of the oligonucleotide, and in particular embodimentsaromatic groups may be attached to each of the 3′- and 5′-positions. Intheir simplest form, the compounds of the invention can be representedby the formula:(A—L—)_(n)—Owherein A comprises the aromatic group, L is an optional linker group(i.e., a linker or a direct bond), O is an oligonucleotide and n is aninteger between 1 and 1+m where m is the total number of nucleosidescomprising the oligonucleotide. In this formula, the bond — is used inthe conventional manner to indicate a covalent linkage between moietiesat each end of the bond. In preferred embodiments, n=1 or 2 and the Amoiety is attached to the oligonucleotide component at one or each ofthe 3′- and 5′-terminii.

The oligonucleotide components of the compounds typically comprise 2-50nucleosides (i.e., m=2-50), and the intersubunit linkages between thenucleosides can be formed using any compatible chemistry, including, butnot limited to: phosphodiester; phosphotriester; methylphosphonate;P3′→N5′ phosphoramidate; N3′→P5′ phosphoramidate; N3′→P5′thio-phosphoramidate; and phosphorothioate linkages. In preferredembodiments, the sequence of the oligonucleotide O is selected such thatit is complementary to the template region of the RNA component oftelomerase.

In particular embodiments, the covalently linked aromatic moiety A is apyrimidine or purine nucleobase or an analog or derivative thereof.Particular examples include guanine, cytosine, thymine, uracil andadenine. In other embodiments, the covalently linked aromatic moiety Ais polyaromatic substituted or unsubstituted hydrocarbon such as anintercalator, reporter molecule, chromophore or fluorophore. Particularexamples include trityl-based groups, fluoresceins, rhodamines,coumarins, acridines, and anthraquinones. One or more A moieties may beattached to the oligonucleotide at the 3′- or 5′-terminus or at anintermediate location on the oligonucleotide. The A moiety may beattached (with or without a linker) to any compatible group on theoligonucleotide, including to the sugar ring, the internucleosidelinkage, and the base. In conjugates having more than one A moietyattached to the oligonucleotide, each A moiety, each linker, and eachsite of attachment may be independently chosen. In certain embodimentsinvolving conjugates having more than one A moiety, a first A moiety iscovalently linked to the oligonucleotide O, and other A groups arecovalently linked to the first A moiety, optionally through a linkergroup.

A variety of linkers (L) may be used to covalently link the A moiety tothe oligonucleotide O, or sequentially join A moieties, as describedherein. Where A is selected to be a nucleobase, it is preferred that thelinker L be a relatively flexible linker so as to permit movement of theA moiety relative to the oligonucleotide O or to sequentially joinedother A moieties. Where no linker is required, L is a direct bondbetween A and O.

The oligonucleotide conjugate compounds of the present invention may beused in methods to inhibit telomerase enzymatic activity. Such methodscomprise contacting a telomerase enzyme with a compound of theinvention. The oligonucleotide conjugate compounds of the presentinvention may also be used to inhibit telomerase in cells that expresstelomerase, thereby inhibiting the proliferation of such cells. Suchmethods comprise contacting a cell or cells having telomerase activitywith a compound of the invention. Cells treated in this manner, whichmay be cells in vitro, or cells in vivo, will undergo telomereshortening and cease proliferating. Since cancer cells requiretelomerase activity to proliferate, the compounds of the invention areparticularly useful for inhibiting the growth of cancer cells, and maybe used in therapeutic applications to treat cancer.

Aspects of the invention therefore include oligonucleotide conjugatecompounds as described herein for use in medicine, and in particular foruse in treating cancer.

Also provided herein are pharmaceutical compositions comprising anoligonucleotide conjugate according to the invention formulated in apharmaceutically acceptable excipient.

Two oligonucleotide conjugates that are illustrative of the compounds ofthe present invention are shown schematically below, with the threecomponents A, L and O shown as bracketed. In Illustration A, the two3′-nucleosides of the oligonucleotide (O) are shown, and the aromaticgroup (A) fluorescein is linked to the 3′-end of the oligonucleotidethrough a linker (L, a thiourea group) attached to the sugar ring of the3′ terminal nucleoside. In Illustration B, the two 3′-nucleosides of theoligonucleotide (O) are shown, and the aromatic group (A) guanine islinked to the 3′-end of the oligonucleotide through a linker (L, an opensugar ring) attached to the thio-phosphoramidate internucleoside linkageof the 3′ terminal nucleoside. In each illustration, B represents thenucleoside bases of the oligonucleotide, and the oligonucleosidebackbone linkages shown are N3′→P5′ thio-phosphoramidate linkages.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of an exemplary oligonucleotide conjugate ofthe invention wherein the polynucleotide AGG (SEQ ID NO.: 1) havingphosphoramidate internucleoside linkages is attached to fluorescein viaa thiourea group.

FIG. 2 shows the structure of an exemplary oligonucleotide conjugate ofthe invention wherein the polynucleotide AGG (SEQ ID NO:1) havingthiophosphoramidate internucleoside linkages is attached to guanine viaa Type 3 Linker (specifically, an open-ring sugar linker, ORS2).

FIG. 3 shows the structures of exemplary aromatic systems having linkergroups attached to them.

DETAILED DESCRIPTION A. Definitions

An “alkyl group” refers to an alkyl or substituted alkyl group having 1to 20 carbon atoms, such as methyl, ethyl, propyl, and the like. Loweralkyl typically refers to C₁ to C₅. Intermediate alkyl typically refersto C₆ to C₁₀. An “acyl group” refers to a group having the structure RCOwherein R is an alkyl. A lower acyl is an acyl wherein R is a loweralkyl.

An “alkylamine” group refers to an alkyl group with an attachednitrogen, e.g., 1-methyl1-butylamine (CH₃CHNH₂CH₂CH₂CH₃).

An “aryl group” refers to an aromatic ring group having 5-20 carbonatoms, such as phenyl, naphthyl, anthryl, or substituted aryl groups,such as, alkyl- or aryl-substitutions like tolyl, ethylphenyl,biphenylyl, etc. Also included are heterocyclic aromatic ring groupshaving one or more nitrogen, oxygen, or sulfur atoms in the ring.

“Oligonucleotide” refers to nucleoside subunit polymers having betweenabout 2 and about 50 contiguous subunits. The nucleoside subunits can bejoined by a variety of intersubunit linkages, including, but not limitedto, phosphodiester, phosphotriester, methylphosphonate, P3′→N5′phosphoramidate, N3′→P5′ phosphoramidate, N3′→P5′ thio-phosphoramidate,and phosphorothioate linkages. Further, “oligonucleotides” includesmodifications, known to one skilled in the art, to the sugar backbone(e.g., ribose or deoxyribose subunits), the sugar (e.g., 2′substitutions), the base, and the 3′ and 5′ termini. In embodimentswhere the oligonucleotide moiety includes a plurality of intersubunitlinkages, each linkage may be formed using the same chemistry or amixture of linkage chemistries may be used. The term “polynucleotide”,as used herein, has the same meaning as “oligonucleotide” and is usedinterchangeably with “oligonucleotide”.

Whenever an oligonucleotide is represented by a sequence of letters,such as “ATGUCCTG,” it will be understood that the nucleotides are in5′→3′ order from left to right. Representation of the base sequence ofthe oligonucleotide in this manner does not imply the use of anyparticular type of internucleoside subunit in the oligonucleotide.

As used herein, “nucleoside” includes the natural nucleosides, including2′-deoxy and 2′-hydroxyl forms, e.g. as described in Komberg and Baker,DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992), and analogs.“Analogs” in reference to nucleosides includes synthetic nucleosideshaving modified base moieties and/or modified sugar moieties, e.g.described generally by Scheit, Nucleotide Analogs (John Wiley, New York,1980). Such analogs include synthetic nucleosides designed to enhancebinding properties, e.g. stability, specificity, or the like, such asdisclosed by Uhlmann and Peyman (Chemical Reviews, 90:543-584, 1990).

A “nucleobase” is defined herein to include (i) typical DNA and RNAnucleobases (uracil, thymine, adenine, guanine, and cytosine), (ii)modified nucleobases or nucleobase analogs (e.g., 5-methyl-cytosine,5-bromouracil, or inosine) and (iii) nucleobase analogs. A nucleobaseanalog is a chemical whose molecular structure mimics that of a typicalDNA or RNA base.

As used herein, “pyrimidine” means the pyrimidines occurring in naturalnucleosides, including cytosine, thymine, and uracil, and analogsthereof, such as those containing oxy, methyl, propynyl, methoxy,hydroxyl, amino, thio, halo, and like, substituents. The term as usedherein further includes pyrimidines with protection groups attached,such as N₄-benzoylcytosine. Further pyrimidine protection groups aredisclosed by Beaucage and Iyer (Tetrahedron 48:223-2311, 1992).

As used herein, “purine” means the purines occurring in naturalnucleosides, including adenine, guanine, and hypoxanthine, and analogsthereof, such as those containing oxy, methyl, propynyl, methoxy,hydroxyl, amino, thio, halo, and like, substituents. The term as usedherein further includes purines with protection groups attached, such asN₂-benzoylguanine, N₂-isobutyrylguanine, N₆-benzoyladenine, and thelike. Further purine protection groups are disclosed by Beaucage andIyer (cited above).

As used herein, the term “protected” as a component of a chemical namerefers to art-recognized protection groups for a particular moiety of acompound, e.g. “5′-protected-hydroxyl” in reference to a nucleosideincludes triphenylmethyl (i.e., trityl), p-anisyldiphenylmethyl (i.e.,monomethoxytrityl or MMT), di-p-anisylphenylmethyl (i.e.,dimethoxytrityl or DMT), and the like. Art-recognized protection groupsinclude those described in the following references: Gait, editor,Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford,1984); Amarnath and Broom, Chemical Reviews, 77:183-217, 1977; Pon etal., Biotechniques, 6:768-775, 1988; Ohtsuka et al., Nucleic AcidsResearch, 10:6553-6570, 1982; Eckstein, editor, Oligonucleotides andAnalogues: A Practical Approach (IRL Press, Oxford, 1991), Greene andWuts, Protective Groups in Organic Synthesis, Second Edition, (JohnWiley & Sons, New York, 1991), Narang, editor, Synthesis andApplications of DNA and RNA (Academic Press, New York, 1987), Beaucageand Iyer (cited above), and like references.

The term “halogen” or “halo” is used in its conventional sense to referto a chloro, bromo, fluoro or iodo substituent. In the compoundsdescribed and claimed herein, halogen substituents are generally fluoro,bromo, or chloro, preferably fluoro or chloro.

B. Design and Synthesis of the Oligonucleotide Conjugates

The present invention is directed generally to compounds having theformula:(A—L—)_(n)—O

wherein A is an aromatic group, L is an optional linker (i.e. a linkeror a direct bond), O is an oligonucleotide, n is an integer between 1and 1+m where m is the total number of nucleosides comprising theoligonucleotide. Design of the compounds therefore requires theselection of three entities, A, L and O and the determination of thestructural linkages between these entities.

Selection of O

The oligonucleotide sequence of O is selected such that it iscomplementary to the RNA sequence of the target telomerase, e.g., humantelomerase. The RNA component of human telomerase has been sequenced(see Feng et al., Science 269 (5228), 1236-1241, 1995, sequence dataalso available on GenBank, Accession No. U86046). Within the humantelomerase RNA (“hTR”) sequence is a region identified as the “templateregion” that functions to specify the sequence of the telomeric repeatsthat telomerase adds to the chromosome ends; this template region isessential to the activity of the telomerase enzyme (see Chen et al.,Cell 100:503-514, 2000; Kim et al, Proc. Nat'l. Acad. Sci, USA 98(14):7982-7987, 2001). The template region is an 11 nucleotide region ofsequence 5′-CUAACCCUAAC-3′ (SEQ ID: 2) that has therefore beenidentified as a particularly suitable target for inhibitoryoligonucleotides. Accordingly, while the selection of theoligonucleotide sequence of the O component of the oligonucleotideconjugates can be made from any region of the hTR sequence, selection ofa sequence complementary to the template and/or adjacent regions of thehTR sequence is preferred. The sequence selected is preferably exactlycomplementary to the corresponding hTR sequence. While mismatches may betolerated in certain instances, they are expected to decrease thespecificity and activity of the resultant oligonucleotide conjugate. Inparticular embodiments, the base sequence of the oligonucleotide O isthus selected to include a sequence of from 2 to 11 nucleotides exactlycomplementary to the template region of human telomerase RNA. Optimaltelomerase inhibitory activity may be obtained when the full length ofthe oligonucleotide O is selected to be complementary to a part of thehTR template region sequence.

The length of the oligonucleotide O may vary from 2 to about 50nucleosides. However, oligonucleotides of shorter lengths are preferredsince shorter molecules are (a) more easily synthesized and may beproduced at a lower cost and (b) likely to be more bio-available. Thus,in preferred embodiments the length of the oligonucleotide O componentsis from 4-15 nucleosides, and in particular embodiments from 4-8nucleosides. The examples of oligonucleotide conjugates presented belowshow that lengths of O of 4-8 nucleosides have potent telomeraseinhibition activity. Oligonucleotide conjugates of the invention includethose having oligonucleotide (O) components containing 2, 3, 4, 5, 6, 7,8, 9, and 10 or more nucleosides. One of skill in the art willappreciate that the selection of the sequence and length of the Ocomponent may also be affected by the selection of the L and Acomponents of the conjugate. Simple experimentation using the standardtelomerase activity assays described herein will facilitate selection ofthe optimal combinations. Preferably, the oligonucleotide conjugateswill include a region of from 2 to 11 consecutive bases having asequence complementary to all or part of the template region of humantelomerase RNA. Illustrative oligonucleotide sequences that may be usedin the conjugates of the invention include: TAGGGTTAGACAA (SEQ ID: 3);GTTAGGGTTAG (SEQ ID: 4); AGGGTTAG (SEQ ID: 5); GGGTTAG (SEQ ID: 6);GTTAGG (SEQ ID: 7); TTAGGG (SEQ ID: 8); TTA (SEQ ID: 9); AGGG (SEQ ID:10); GGGTTA (SEQ ID: 11); TGAGTG (SEQ ID: 12); and GTAGGT (SEQ ID: 13).

The choice of the type of inter-nucleoside linkages used in thesynthesis of the O component may be made from any of the availableoligonucleotide chemistries, including but not limited to,phosphodiester, phosphotriester, methylphosphonate, P3′→N5′phosphoramidate, N3′→P5′ phosphoramidate, N3′→P5′ thio-phosphoramidate,and phosphorothioate linkages. Oligonucleotides having at least oneN3′→P5′ phosphoramidate or N3′→P5′ thio-phosphoramidate linkage may havecharacteristics that provide advantages in the therapeutic context.Exemplary oligonucleotide linkages may be represented by the formula:3′-[-Z₆—P(═O)(—Z₇R)—O-]-5′

wherein Z₆ is O or NH; Z₇ is O or S; and R is selected from the groupconsisting of hydrogen, alkyl, aryl and salts thereof.

Methods for synthesizing oligonucleotides suitable for incorporationinto the conjugates described herein are well known in the art. By wayof example, oligonucleotide N3′→P5′ thiophosphoramidates for use in theinvention may be synthesized using the phosphoramidite transfermethodology of Pongracz et al., Tet. Let. 40: 7661-7664, 1999). Thissynthetic strategy employs 3′-NH-trityl-protected 3′-aminonucleoside5′-O-cyanoethyl-N,N-diisopropylaminophosphor-amidites which areprocessed through the steps of 1) detritylation, 2) coupling; 3)capping; 4) sulfurization. To achieve a step-wise sulfurization of theinternucleaside phosphoramidite group formed after the coupling step,sulfurizing agents such as elemental sulfur S₈ or PADS(diphenylacetyldisulfide) or the commonly used Beaucagereagent-3H-1,2-benzodithiol-3-one 1,1 dioxide (Iyer et al., J. OrganicChemistry 55:4693-4699, 1990) can be used. The oligonucleotide synthesesmay be performed (1 μmole synthesis scale) with a 1% solution ofBeaucage reagent in anhydrous acetonitrile or 15% S₈ in CS₂/Et₃N, 99/1(vol/vol) as the sulfurizing agent.

Chimeric N3′→P5′ phosphoramidate-phosphorthioamidate oligonucleotidescan be made by using an oxidation step(s) after the coupling step, whichresults in formation of a phosphoramidate internucleoside group.Similarly, phosphodiester-phosphorthioamidates can be made by using5′-phosphoramidite-3′-O-DMTr-protected nucleotides as monomeric buildingblocks. These synthetic approaches are known in the art.

Selection of A

It is believed that the conjugates of the present invention bind totelomerase in two aspects, thereby inhibiting function of the enzyme.The first aspect of the binding is the base-pair complementarity betweenthe nucleosides of hTR and the nucleosides of the oligonucleotide (O)component of the conjugate. The second aspect of the binding is theinteraction between the telomerase protein component and the A componentof the conjugate. A wide range of substituents may serve this secondbinding aspect provided by the A component which, as a matter ofconvenience, is referred to herein as an “aromatic” moiety. In thiscontext, the term “aromatic” is used broadly to encompass, for example,polyaromatic hydrocarbons, mono- and poly-cyclic heteroaromatic systemsand nucleobases (including modified nucleobases and nucleobase analogs).

Classes of suitable aromatic systems include intercalators, reportermolecules, chromophores, fluorophores and nucleobases (includingnucleobase derivatives and nucleobase analogs). Particular examples ofsuitable A substituents include fluoresceins, rhodamines, coumarins,acridines, triphenyl carbons, anthraquinones and purine and pyrimidinenucleobases. Where the A moiety is selected to be a nucleobase,nucleobases capable of hydrogen-bonding are preferred, sincehydrogen-bonding is expected to increase the stability of theinteraction between the A moiety and the protein component oftelomerase. Examples of nucleobases capable of hydrogen-bonding includeadenine, guanine, cytosine, thymine and uracil.

Illustrative fluorophores are shown below:

where X, X₁ and X₂ are independently selected from the group consistingof O, S, and N; X₃ is hydrogen, halogen, or alkyl; X₄ is C, N, O, or S;R₁ and R₂ are independently selected from the group consisting of H,methyl, ethyl, and propyl; and R₃, R₄ and R₅ are independently selectedfrom the group consisting of H, hydroxyl, halogen, alkyl, aryl,carboxyl, and X-alkyl, and

is a single or a double bond.

Particular examples of suitable aromatic systems are fluorescein, havingthe formula:

N,N′-tetramethylrhodamine, having the formula:

Trityl having the formula:

And pyrene-butyric acid, having the formula:

Examples of suitable A moieties which fall into the nucleobase class ofA substituents include the nucleobases found in DNA and RNA,specifically the pyrimidines cytosine, thymine and uracil, and thepurines adenine and guanine, the structures of which are well known. Awide range of modified nucleobases and nucleobase analogs are known inthe art; widely available forms include halogen-, nitro- andhydroxy-substituted nucleobases, preferably substituted at the 2-, 6- or8-positions, such as 2-chloroadenine, 6-thioguanine, 6-chloroguanine,8-bromoadenine, 8-iodoadenine, 8-hydroxyguanine and 8-nitroguanine, andnucleobases with ring substitutions such as deaza nucleobases in whichone or more nitrogen atoms in the heterocyclic ring is substituted for acarbon. Examples of ring-substituted nucleobase derivatives include2,6-diaminopurines, 7-alkylpurines, 7-alkynylpurines and 7-deazapurines.When the A substitutent is selected to be a nucleobase, nucleobaseanalog or nucleobase derivative, it is preferred that the nucleobase becapable of hydrogen-bonding, thereby increasing the stability of theinteraction between the A moiety and the protein component oftelomerase.

Other aromatic substituents (shown conjugated with linker (L) groups)are depicted in FIG. 3. “A” substituents that may be employed in theoligonucleotide conjugates of the present invention may be purchasedfrom chemical reagent suppliers or synthesized using standard chemicalsynthesis procedures. As with the selection of the oligonucleotide (O)component, the activity of oligonucleotide conjugates having various Amoieties may readily be determined using standard telomerase activityassays.

Selection of L

The linkage between the O and A components may be facilitated by theincorporation of a linker sequence. Such a sequence may serve not onlyto facilitate the chemical conjugation of the two moieties, but may alsoserve to increase the activity of the conjugate by conferringflexibility and thereby enhancing the availability of each moiety tobind to its target on the telomerase enzyme. Linkers may be conjugateddirectly to the sugar ring of the oligonucleotide O, or to theinternucleoside backbone, such as the phosphodiester or phosphoramidateinternucleoside linkages, or to the nucleobase of the oligonucleotide. Avariety of linker groups may be utilized, depending on the selection ofthe A component. Exemplary linkers are the structures:

Wherein:

Z₁, Z₂, and Z₃ are independently selected from the group consisting ofO, S, and NR₄, wherein

R₄ is H or lower alkyl and Z₁ and Z₃ can be direct bonds;

Z₄ is O or NH;

Z₅ is OR′, SR′, or methyl wherein R′ is selected from the groupconsisting of hydrogen, alkyl, aryl and salts thereof;

R₉ is H, halogen or lower alkyl, and m2 is an integer from 0 to 10.

In instances where no linker group is desired, L is a direct bondbetween A and O.

One example of a Type 1 linker is the thiourea linker in which: Z₁=NH;Z₂=S; Z₃=NH; and m2=0 (i.e., there is no carbon between the Z₃=Nsubstituent and the A moiety. A Type 1 thiourea linker is shown in thestructure depicted in Illustration A in the Summary section above.

A representation of the structure of oligonucleotide conjugates having aType 2 linker is shown below:

wherein each B is a base independently selected to be a purine orpyrimidine or an analog thereof such as uracil, thymine, adenine,guanine, cytosine, 5-methylcytosine, 5-bromouracil and inosine, A is thearomatic moiety and the linker shown linking the 3′ sugar ring of O tothe A moiety is a type 2 linker. From this arrangement, it is apparentthat the definition of the Type 2 linker structure incorporatescomponents of an internucleoside linkage. Reference to a “C5” or “C6”linker refers to a Type 1 or 2 linker in which m2=5 or 6.

Where the A moiety is a nucleobase, a preferred type of linker is theType 3 linker, which may be conjugated directly to the 3′ position ofthe 3′ sugar ring of the oligonucleotide O:

Wherein:

X=N or O

Y=O or S

Z=O or S

W=N, O, S or lower alkyl (preferably CH₂)

V is lower alkyl

Q=O, S or NR′″, wherein R′″ is H, lower alkyl or lower acyl

R′ and R″ are independently ═H, OH, alkyl (including substituted alkyl,preferably lower alkyl) or alkylamine.

Typically, a Type 3 linker is conjugated to the sugar ring of the 3′nucleobase of the oligonucleotide O.

Particularly preferred forms of the Type 3 linker are linkers thatinclude phosphate groups conjugated to open ring sugars, thus resemblinga modified oligonucleotide phosphate-sugar backbone. Examples of Type 3linkers incorporating open ring sugar groups are Open Ring Sugar Linkers1 and 2, which are illustrated below and are in the examples shown inTable 2 below:

(As noted above, the Y substituent may be oxygen or sulfur and may beselected such that this substituent is consistent with the equivalentsubstituent in the internucleoside linkages of the oligonucleotide O.Thus, when O is selected to be a phosphoramidate oligonucleotide, Y maybe selected to be oxygen, and when O is selected to be athio-phosphoramidate oligonucleotide, Y may be selected to be sulfur.)

Notably, where the A moiety is selected to be guanine and the L moietyis Open Ring Sugar Linker 1 or 2, this combination produces acyclovir organciclovir, respectively, conjugated to the phosphate group, which inturn is then conjugated to the 3′ sugar ring of the oligonucleotide O.

It will be apparent that another way of representing the linkageproduced by linking a nucleobase to the 3′ sugar of the oligonucleotideO using a Type 3 linker is to represent the phosphate group not as partof the Type 3 linker, but instead as the 3′ internucleoside linkage ofthe oligonucleotide. In this instance, the structure may be representedas follows, with the modified Type 3 linker now referred to as a Type 4linker, with substituents as described for the Type 3 linker above:

In oligonucleotide conjugates of the invention represented by theformula (A—L—)_(n)—O, when A is selected to be a nucleobase, the formulais not intended to include the possibility that A—L simply represents anadditional conventional nucleoside or nucleotide that is part of theoligonucleotide O. Rather, A—L represents a moiety that distinguishesthe oligonucleotide conjugates of the invention both in structure and infunction from conventional known oligonucleotides.

Thus, in particular embodiments, when A is a nucleobase, L should notinclude a closed ring sugar group, of the type that is found innucleobase linkages in conventional oligonucleotides. In part, thisreflects the preference that the linker L in conjugates in which A is anucleobase should be a flexible linker; nucleobase linkages employingclosed ring sugar groups used in conventional oligonucleotides are notconsidered to produce flexible linkers. As used herein the phrase“closed ring sugar group” includes heterocyclic 5 and 6 member closedring sugar groups described in the art for use as nucleobase linkages inoligonucleotides, including ribose and arabinose and known derivativesof these sugars which include a 5 or 6 member closed ring, such asribose, deoxyribose, 2′-O methylribose and 2′-O fluororibose.

In other embodiments, the oligonucleotide conjugates of the inventionare distinguished from conventional oligonucleotides in that the linkerL is selected so that it is not the same as one or more inter-nucleobaselinkages found in the oligonucleotide O. In this context, it will beappreciated that reference to the inter-nucleobase linkages found in theoligonucleotide O encompasses both the sugar moiety and the associatedphosphate-based moiety that join the nucleobases of the oligonucleotide.Thus, in this embodiment, the oligonucleotide conjugates of theinvention may be represented as (A—L—)_(n)—O, wherein A is a nucleobase,n is an integer between 1 and m+1, m is the total number of nucleosidesin O, O is an oligonucleotide that comprises m nucleobases and m−1inter-nucleobase linkages L′, and L is a linker that is not the same asany L′ found in the oligonucleotide O. An example of such a conjugate inwhich m=4 is as follows: A—L—B¹—L′—B²—L′—B³—L′—B⁴ wherein A is anucleobase, B¹, B², B³ and B⁴ are independently selected nucleobases, Land L′ are linkers, wherein L is not the same as L′.

It is important to note that many different linkers may be used in thepresent invention to join aromatic A groups to the oligonucleotide groupO. The foregoing linkers are illustrative only.

Arrangement of Components

As implied by the structure (A—L—)_(n)—O, wherein A is an aromaticgroup, L is an optional linker, O is an oligonucleotide, n is an integerbetween 1 and 1+m where m is the total number of nucleosides comprisingthe oligonucleotide, multiple A substituents may be conjugated to theoligonucleotide. Moreover, the A substituents may be attached to theoligonucleotide, optionally through a linker L, at the 3′-position, atthe 5′-position, on the intersubunit linkage, on the sugar ring, on thebase, or combinations thereof. However, in typical embodiments, one ortwo A substituents are used (i.e., n=1 or 2), and these are generallyattached (through L) to the 3′-position or the 5′-position of theterminal sugar residue of the oligonucleotide O, or to the intersubunitlinkage that is attached to the sugar at that point. When A is selectedto be a nucleobase, it is preferably conjugated to the 3′ terminus ofthe oligonucleotide O in order to enhance telomerase inhibitoryactivity, and most preferably, is conjugated, via a linker L, to the 3′position of the sugar of the 3′ terminal nucleoside.

FIG. 1 illustrates an example in which the A substituent is fluoresceinattached to the 3′-position of the oligonucleotide via a thiourea linkerL. FIG. 2 illustrates an example in which the A substituent is guanineattached to the 3′ terminal intersubunit linkage of the oligonucleotidevia an open ring sugar linker L. Where two or more A substituents areutilized, the A substituents are independently selected. As with theselection of all of the components, the design of the conjugate canreadily be tested by performing standard telomerase activity assays asdescribed below.

In particular embodiments, the integer n is selected to be 1, such thatthe structure of the oligonucleotide conjugate can be represented as:A—L—O

and wherein 0 is preferably an oligonucleotide that comprises a sequenceof from 2 to 11 nucleotides exactly complementary to a sequence withinthe template region of human telomerase RNA.

In such embodiments, when A is selected to be a nucleobase, it ispreferably conjugated to the 3′ position of the 3′ terminal sugar of theoligonucleotide O. While various chemistries may be employed for theinternucleoside linkages within O, phosphorothioate, phosphoramidate,and thiophosphoramidate linkages are particularly desirable. Telomeraseinhibition activity may be particularly good when the sequence of theoligonucleotide O is selected such that it has a 3′ terminus of GGG,since oligonucleotides of this type may form highly stable interactionswith the telomerase enzyme. Exemplary oligonucleotide sequences includeTTAGGG (SEQ ID: 8) and AGGG (SEQ ID: 10). Selection of a flexiblelinker, such as a Type 3 linker, preferably a Open Sugar Ring Linker 1or Open Sugar Ring Linker 2 may also enhance telomerase inhibitoryactivity, as may the selection of a purine nucleobase, in particularguanine, as the A moiety.

In another embodiment of the invention, where the oligonucleotideconjugate (A—L)_(n)—O is designed so that n>1, a first selected Asubstituent is covalently conjugated to the oligonucleotide O, and otherA substituents are sequentially linked, optionally through a linker, tothe first A substituent. An example of such a conjugate isTAA-ORS2-G*G*G* as shown in Table 2, in which a first guanine nucleobaseis linked through a Type 3 linker (specifically an Open Ring SugarLinker 2) to the 3′ position of the 3′ terminal sugar ring of theoligonucleotide O. A second guanine nucleobase is then linked to thefirst, also through an Open Ring Sugar Linker 2, and the third is thenlinked to the second in a similar manner.

Synthesis of the Oligonucleotide Conjugates

The aromatic substituent can be covalently linked to the oligonucleotideusing standard chemical syntheses. Typically, the nucleophilic sites onthe oligonucleotide are first protected and the desired site ofattachment is selectively deprotected. The oligonucleotide is thenreacted with, for example in the case of the aromatic substituent beingfluorescein, fluorescein containing a reactive group such asisothiocyanate, isocyanate, monochlorotriazine, dichlorotriazine, mono-or di-halogen substituted pyridine, mono- or di-halogen substituteddiazine, maleimide, aziridine, sulfonyl halide, acid halide,hydroxysuccinimide ester, hydroxysulfosuccinimide ester, imido ester,hydrazine, azidonitrophenyl, azide, 3-(2-pyridyl dithio)-proprionamide,glyoxal or aldehyde, to yield a modified oligonucleotide in whichfluorescein is bound to the oligonucleotide, preferably through athiourea or an amide group, as shown in the scheme below:

The linker L may be joined to the aromatic system A at position 1, 2, 3,or 4. Preferably, L is joined to A at the 3 position.

Alternatively, the linker can first be converted into a phosphoramiditefollowed by coupling to the oligonucleotide, as described in U.S. Pat.No. 5,583,236 to Brush. Typically, when the aromatic system isfluorescein, commercially available fluorescein 3-isothiocyanatediacetate is reacted sequentially with aminopenta-6-ol and then aphosphitylating reagent to yield a phosphoramidite linked to fluoresceinvia a five carbon chain and a thiourea group, as shown below:

Some examples of a linker L joined to a suitable aromatic system A areillustrated in FIG. 3.

Where the A moiety is a nucleobase, the same general syntheticapproaches are employed. Nucleobases may be purchased with groupsalready attached that are suitable as linkers or which may be readilymodified to produce suitable linkers. For example, guanine mayconveniently be purchased with attached open ring sugars in the form ofacyclovir or ganciclovir, in which the conjugated open ring sugar formsa Type 4 linker as shown above. The open ring sugar is thenphosphitylated to form a phosphoramidate (with other reactive groupspresent in the linker being protected with an appropriate protectinggroup). The resultant phosphoramidate is then coupled to theoligonucleotide using standard procedures on an automated DNAsynthesizer, as described above. Subsequent modification of the linker(e.g., sulfurization to produce a thiophosphoramidate moiety) isperformed using standard chemistry.

Telomerase Inhibition Assays

The conjugates of the present invention may be used to inhibit or reducetelomerase enzyme activity and/or proliferation of cells havingtelomerase activity. In these contexts, inhibition or reduction of theenzyme activity or cell proliferation refer to a lower level of themeasured activity relative to a control experiment in which the enzymeor cells are not treated with the conjugate. In particular embodiments,the inhibition or reduction in the measured activity is at least a 10%reduction or inhibition. One of skill in the art will appreciate thatreduction or inhibition of the measured activity of at least 20%, 50%,75%, 90% or 100% may be preferred for particular applications.

Methods for measuring telomerase activity, and the use of such methodsto determine the telomerase inhibitory activity of compounds are wellknown. For example, the TRAP assay is a standard assay method formeasuring telomerase activity in a cell extract system and has beenwidely used in the search for telomerase inhibiting compounds (Kim etal., Science 266:2011, 1997; Weinrich et al., Nature Genetics 17:498,1997). The TRAP assay measures the amount of radioactive nucleotidesincorporated into elongation products (polynucleotides) formed bynucleotide addition to a telomerase substrate or primer. Theradioactivity incorporated can be measured as the intensity of a band ona detection screen (e.g. a Phosphorimager screen) exposed to a gel onwhich the radioactive products are separated. The TRAP assay is alsodescribed in detail in U.S. Pat. Nos. 5,629,154, 5,837,453 and5,863,726, and its use in testing the activity of telomerase inhibitorycompounds is described in various publications including WO 01/18015. Inaddition, the following kits are available commercially for researchpurposes for measuring telomerase activity: TRAPeze® XK TelomeraseDetection Kit (Cat. s7707; Intergen Co., Purchase N.Y.); and TeloTAGGGTelomerase PCR ELISA plus (Cat. 2,013,89; Roche Diagnostics,Indianapolis Ind.).

Typically, the ability of the conjugates of the invention to inhibittelomerase activity will first be confirmed by performing a biochemicaltelomerase activity assay, such as the TRAP assay, and comparing theresults obtained in the presence of the conjugate with results obtainedfrom a control experiment performed without the conjugate. Such datapermit the calculation of an IC₅₀ value for the conjugate (theconcentration of the test compound at which the observed activity for asample preparation is observed to fall one-half of its original or acontrol value). Using such methods, IC₅₀ values for several of theoligonucleotide conjugates of the present invention were determined, andfound to be below 10 μM.

In addition, the specificity of the oligonucleotide conjugates fortelomerase RNA can be determined by performing hybridization tests withand comparing their activity (IC₅₀) with respect to telomerase and toother enzymes known to have essential RNA components, such asribonuclease P. Compounds having lower IC₅₀ values for telomerase ascompared to the IC₅₀ values toward the other enzymes being screened aresaid to possess specificity for telomerase.

Having confirmed that a particular conjugate is an effective telomeraseinhibitor using a biochemical assay, it may then be desirable toascertain the ability of the conjugate to inhibit telomerase activity incells. Oligonucleotide conjugates that effectively inhibit telomeraseactivity in cells will, like other known telomerase-inhibitingcompounds, induce crisis in telomerase-positive cell lines. Cell linesthat are suitable for such assays include HME50-5E human breastepithelial cells, and the ovarian tumor cell lines OVCAR-5 and SK-OV-3.Importantly however, in normal human cells used as a control, such as BJcells of fibroblast origin, the observed reduction in telomere length isexpected to be no different from untreated cells. In selecting anoligonucleotide conjugate of the invention for therapeutic applications,one would typically select a conjugate that produced no significantcytotoxic effects at concentrations below about 20 μM in normal cells.Testing the ability of a candidate compound to inhibit telomerase incell-based assays is also routine, and is described in WO 01/18015,Herbert et al., Oncogene, 21: 638-642, 2002; Gryaznov et al.,Nucleosides, Nucleotides and Nucleic Acids, 20: 401-410, 2001; Izbika etal., Cancer Res., 59:639-644, 1999; Shammas & Corey Oncogene 18:6191-6200, 1999; and Pitts & Corey, Porc. Nat'l. Acad. Sci. USA,95:11549-11554, 1998.

Confirmation of the ability of an oligonucleotide conjugate to inhibittumor cell growth in vivo can be obtained using established xenograftmodels of human tumors, in which the test conjugate is administeredeither directly to the tumor site or systemically, and the growth of thetumor is followed by physical measurement. Animals treated witholigonucleotide conjugates of the invention are expected to have tumormasses that, on average, may increase for a period following the initialdosing, but will begin to shrink in mass with continuing treatment. Incontrast, untreated control mice are expected to have tumor masses thatcontinue to increase. Examples of such xenograft models in screening forcancer therapeutics are described in Scorski et al., Proc. Nat'l. Acad.Sci. USA, 94: 3966-3971, 1997; and Damm et a., EMBO J., 20:6958-6968,2001.

C. Formulation of Oligonucleotide Conjugates

The present invention provides oligonucleotide conjugates that canspecifically and potently inhibit telomerase activity, and which maytherefore be used to inhibit the proliferation of telomerase-positivecells, such as tumor cells. A very wide variety of cancer cells havebeen shown to be telomerase-positive, including cells from cancer of theskin, connective tissue, adipose, breast, lung, stomach, pancreas,ovary, cervix, uterus, kidney, bladder, colon, prostate, central nervoussystem (CNS), retina and circulating tumors (such as leukemia andlymphoma). Accordingly, the oligonucleotide conjugates provided hereinmay provide a broadly useful in treating a wide range of malignancies.More importantly, the oligonucleotide conjugates of the presentinvention can be effective in providing treatments that discriminatebetween malignant and normal cells to a high degree, avoiding many ofthe deleterious side-effects present with most current chemotherapeuticregimes which rely on agents that kill dividing cells indiscriminately.One aspect of the invention therefore is a method of treating cancer ina patient, comprising administering to the patient a therapeuticallyeffective dose of an oligonucleotide conjugate of the present invention.Telomerase inhibitors, including oligonucleotide conjugates of theinvention, may be employed in conjunction with other cancer treatmentapproaches, including surgical removal of primary tumors,chemotherapeutic agents and radiation treatment.

For therapeutic application, an oligonucleotide conjugate of theinvention will be formulated in a therapeutically effective amount witha pharmaceutically acceptable carrier. One or more oligonucleotideconjugates (having different base sequences or linkages) may be includedin any given formulation. The pharmaceutical carrier may be solid orliquid. Liquid carriers can be used in the preparation of solutions,emulsions, suspensions and pressurized compositions. The modifiedoligonucleotides are dissolved or suspended in a pharmaceuticallyacceptable liquid excipient. Suitable examples of liquid carriers forparenteral administration of the oligonucleotides preparations includewater (partially containing additives, e.g., cellulose derivatives,preferably sodium carboxymethyl cellulose solution), alcohols (includingmonohydric alcohols and polyhydric alcohols, e.g., glycols) and theirderivatives, and oils (e.g., fractionated coconut oil and arachis oil).The liquid carrier can contain other suitable pharmaceutical additivesincluding, but not limited to, the following: solubilizers, suspendingagents, emulsifiers, buffers, thickening agents, colors, viscosityregulators, preservatives, stabilizers and osmolarity regulators.

For parenteral administration of the oligonucleotides, the carrier canalso be an oily ester such as ethyl oleate and isopropyl myristate.Sterile carriers are useful in sterile liquid form compositions forparenteral administration.

Sterile liquid pharmaceutical compositions, solutions or suspensions canbe utilized by, for example, intraperitoneal injection, subcutaneousinjection, intravenously, or topically. The oligonucleotides can also beadministered intravascularly or via a vascular stent.

The liquid carrier for pressurized compositions can be halogenatedhydrocarbon or other pharmaceutically acceptable propellant. Suchpressurized compositions may also be lipid encapsulated for delivery viainhalation. For administration by intranasal or intrabronchialinhalation or insufflation, the oligonucleotides may be formulated intoan aqueous or partially aqueous solution, which can then be utilized inthe form of an aerosol.

The oligonucleotide conjugates may be administered topically as asolution, cream, or lotion, by formulation with pharmaceuticallyacceptable vehicles containing the active compound.

The pharmaceutical compositions of this invention may be orallyadministered in any acceptable dosage including, but not limited to,formulations in capsules, tablets, powders or granules, and assuspensions or solutions in water or non-aqueous media. Pharmaceuticalcompositions and/or formulations comprising the oligonucleotides of thepresent invention may include carriers, lubricants, diluents,thickeners, flavoring agents, emulsifiers, dispersing aids or binders.In the case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are required for oral use, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening, flavoring or coloring agents may also be added.

Pharmaceutical compositions and/or formulations comprising theoligonucleotides of the present invention may also include cellular andtissue penetration enhancing agent. Penetration enhancing agents thatmay be employed include, for example, fatty acids, bile salts, chelatingagents, surfactants and non-surfactants (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, 8, 91-192; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). Liposomecarriers may also be used as penetration agents. The use of liposomes tofacilitate cellular uptake is described, for example, in U.S. Pat. No.4,897,355 and U.S. Pat. No. 4,394,448. Numerous publications describethe formulation and preparation of liposomes. Fatty acids and theirderivatives which act as penetration enhancers include, for example,oleic acid, lauric acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,recinleate, monoolein (a.k.a. 1-monooleoyl-rac-glycerol), dilaurin,caprylic acid, arichidonic acid, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, mono- anddi-glycerides and physiologically acceptable salts thereof (i.e.,oleate, laurate, caprate, myristate, palmitate, stearate, linoleate,etc.). Such penetration enhancers may be simply added to theformulation, or may be covalently conjugated to the oligonucleotideconjugates described herein. Methods of conjugating oligonucleotides tolipid moieties are described in, for example, Mishra et al., (1995)Biochemica et Biophysica Acta, 1264: 229-237). The use of syntheticpolymers for the delivery of therapeutic oligonucleotides is describedin Chirila et al. (2002) Biomaterials 23: 321-342. Oligonucleotideconjugates of the invention may thus be further modified by formulationwith a moiety designed to enhance cellular and or tissue penetration (ingeneral terms, a penetration enhancer). In particular embodiments, theoligonucleotide conjugates of the invention may further comprise acovalently linked penetration enhancing agent.

Complex formulations comprising one or more penetration enhancing agentsmay be used. For example, bile salts may be used in combination withfatty acids to make complex formulations. Exemplary combinations includechenodeoxycholic acid (CDCA), generally used at concentrations of about0.5 to 2%, combined with sodium caprate or sodium laurate, generallyused at concentrations of about 0.5 to 5%.

Pharmaceutical compositions and/or formulations comprising theoligonucleotides of the present invention may also include chelatingagents, surfactants and non-surfactants. Chelating agents include, butare not limited to, disodium ethylenediaminetetraacetate (EDTA), citricacid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines). Surfactants include, forexample, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether andpolyoxyethylene-20-cetyl ether; and perfluorochemical emulsions, such asFC43. Non-surfactants include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives, and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone.

Thus, in another aspect of the invention, there is provided a method offormulating a pharmaceutical composition, the method comprisingproviding an oligonucleotide conjugate compound as described herein, andcombining the compound with a pharmaceutically acceptable excipient.Preferably the oligonucleotide conjugate is provided at pharmaceuticalpurity, as defined below. The method may further comprise adding to theoligonucleotide conjugate compound, either before or after the additionof the excipient, a penetration enhancing agent. Exemplary penetrationenhancing agents are listed above. In particular embodiments, thepenetration enhancing agent may be covalently linked to theoligonucleotide conjugate.

The pharmaceutical composition will typically comply with pharmaceuticalpurity standards. For use as an active ingredient in a pharmaceuticalpreparation, an oligonucleotide of this invention is generally purifiedaway from other reactive or potentially immunogenic components presentin the mixture in which they are prepared. Typically, to achievepharmaceutical purity, each active ingredient is provided in at leastabout 90% homogeneity, and more preferably 95% or 99% homogeneity, asdetermined by functional assay, chromatography, or gel electrophoresis.The active ingredient is then compounded into a medicament in accordancewith generally accepted procedures for the preparation of pharmaceuticalpreparations. Thus, in the present invention, providing theoligonucleotide conjugate compounds at pharmaceutical purity requiresthat the compound be provided at at least about 90% homogeneity, andmore preferably at least 95% or 99% homogeneity.

The pharmaceutical composition will also typically be aliquoted andpackaged in either single dose or multi-dose units. The dosagerequirements for treatment with the oligonucleotide conjugates vary withthe particular compositions employed, the route of administration, theseverity of the symptoms presented, the form of the oligonucleotides andthe particular subject being treated.

Pharmaceutical compositions of the invention can be administered to asubject in a formulation and in an amount effective to achieve aclinically desirable result. For the treatment of cancer, desirableresults include reduction in tumor mass (as determined by palpation orimaging; e.g., by radiography, CAT scan, or MRI), reduction in the rateof tumor growth, reduction in the rate of metastasis formation (asdetermined e.g., by histochemical analysis of biopsy specimens),reduction in biochemical markers (including general markers such as ESR,and tumor-specific markers such as serum PSA), and improvement inquality of life (as determined by clinical assessment, e.g., Karnofskyscore).

The amount of oligonucleotide per dose and the number of doses requiredto achieve such effects can be determined empirically using in vitrotests and animal models. An appropriate range for testing can beestimated from the 50% inhibitory concentration determined with isolatedtelomerase or cultured cells. Preparations of isolated telomerase can beobtained according to U.S. Pat. No. 5,968,506. Typically, theformulation and route of administration will provide a localconcentration at the disease site of between 1 μM and 1 nM of theoligonucleotide conjugate.

In general, the oligonucleotide conjugates are administered at aconcentration that affords effective results without causing any harmfulor deleterious side effects. Such a concentration can be achieved byadministration of either a single unit dose, or by the administration ofthe dose divided into convenient subunits at suitable intervalsthroughout the day.

EXAMPLE 1 General Methods

³¹P NMR spectra were obtained on a Varian 400 Mhz spectrometer. ³¹P NMRspectra were referenced against 85% aqueous phosphoric acid. Anionexchange HPLC was performed using a Dionex DX 500 Chromatography System,with a Pharmacia Biotech Mono Q HR 5/5 or 10/16 ion exchange columns.Mass spectral analysis was performed by Mass Consortium, San Diego,Calif. MALDI-TOF analysis of oligonucleotides was obtained using aPerSpective Biosystems Voyager Elite mass spectrometer with delayedextraction. Thermal dissociation experiments were conducted on a CaryBio 100 UV-Vis spectrometer.

All reactions were carried out in oven dried glassware under a nitrogenatmosphere unless otherwise stated. Commercially available DNA synthesisreagents were purchased from Glen Research (Sterling, Va.). Anhydrouspyridine, toluene, dichloromethane, diisopropylethyl amine,triethylamine, acetic anhydride, 1,2-dichloroethane, and dioxane werepurchased from Aldrich (Milwaukee, Wis.).

All non-thio-phosphoramidate oligonucleotides were synthesized on an ABI392 or 394 DNA synthesizer using standard protocols for thephosphoramidite based coupling approach (Caruthers, Acc. Chem. Res.,24:278-284, 1991). The chain assembly cycle for the synthesis ofoligonucleotide phosphoramidates was the following: (i) detritylation,3% trichloroaceticacid in dichloromethane, 1 min; (ii) coupling, 0.1 Mphosphoramidite and 0.45 M tetrazole in acetonitrile, 10 min; (iii)capping, 0.5 M isobutyic anhydride in THF/lutidine, 1/1, v/v, 15 sec;and (iv) oxidation, 0.1 M iodine in THF/pyridine/water, 10/10/1, v/v/v,30 sec.

Chemical steps within the cycle were followed by acetonitrile washingand flushing with dry argon for 0.2-0.4 min. Cleavage from the supportand removal of base and phosphoramidate protecting groups was achievedby treatment with ammonia/EtOH, 3/1, v/v, for 6 h at 55° C. Theoligonucleotides were concentrated to dryness in vacuo after which the2′-t-butyldimethylsilyl groups were removed (if present) by treatmentwith 1M TBAF in THF for 4-16 h at 25° C. The reaction mixtures werediluted with water and filtered through a 0.45 nylon acrodisc (fromGelman Sciences, Ann Arbor, Mich.). Oligonucleotides were then analyzedand purified by IE HPLC and finally desalted using gel filtration on aPharmacia NAP-5 or NAP-25 column. Gradient conditions for IE HPLC:solvent A (10 mM NaOH), solvent B (10 mM NaOH and 1.5 M NaCl); solvent Afor 3 min then a linear gradient 0-80% solvent B within 50 min.

EXAMPLE 2 Synthesis of Oligoribonucleotide N3′→P5′ Phosphoramidates

It was previously reported that homopurine and homopyrimidineoligoribonucleotide N3′→P5′ phosphoramidates could be efficientlyassembled on a solid phase support using a phosphoramidite transferreaction (Gryaznov, et al. (1998) Nucleic Acids Res., 26:4160-4167). Wefound that this methodology works equally well for the synthesis ofheterobased phosphoramidate oligoribonucleotides containing all fournatural bases as well as thymidine and 2,4-diaminopurine (D). Each ofthe prepared oligoribonucleotide N3′→P5′ phosphoramidites weresynthesized starting from the 5′-end using a support-bound2′-deoxy-3′-aminonucleoside as the 5′-terminal residue. Coupling stepsinvolved exchange of the diisopropylamino group of the approaching5′-O-phosphoramidite for the 3′-amino group of the support boundnucleoside. Standard RNA synthesis coupling times (10 min) and activator(1H-tetrazole) were used for each synthetic cycle. Unreacted 3′-aminogroups were then capped with isobutyric anhydride, after which oxidationof the internucleotide phosphoramidite diester linkage into thephosphoramidate group was carried out with aqueous iodine. Subsequentdetritylation of the 3′-amino group of the added residue enabledadditional chain elongation steps to be repeated for the construction ofthe desired oligoribonucleotide phosphoramidates. The resin boundcompounds were then deprotected and cleaved from the support bytreatment with ammonia/ethanol solution. Removal of2′-O-t-butyldimethylsilyl groups was accomplished using 1M TBAF in THFafter which the fully deprotected oligoribonucleotide phosphoramidateswere analyzed and isolated using IE. The mixed base 9-13-meroligoribonucleotide phosphoramidates were synthesized with step-wisecoupling yields of about 96-98%, as judged by MMTr assays. Theoligonucleotides were characterized by both ³¹P NMR and MALDI-TOF massspectra analysis.

EXAMPLE 3 Synthesis of Oligonucleotide N3′→P5′ Thio-Phosphoramidates

Oligonucleotide N3′→P5′ thio-phosphoramidates were prepared by theamidite transfer reaction on an ABI 394 synthesizer. The fully protectedmonomer building blocks were 3′-aminotritylnucleoside-5′-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite wherenucleoside is 3′-deoxythymidine, 2′,3′-dideoxy-N²-isobutyryl-guanosine,2′,3′-dideoxy-N⁶-benzoyl-adenosine or 2′,3′-dideoxy-N⁴-benzoyl-cytidine.5′-Succinyl-3′-aminotrityl-2′,3′-dideoxy nucleosides were coupled withan amino group containing long chain controlled pore glass (LCAA-CPG)and used as the solid support. The synthesis was performed in thedirection of 5′ to 3′. The following protocol was used for the assemblyof oligonucleotide N3′→P5′ thio-phosphoramidates: (i) detritylation, 3%dichloroacetic acid in dichloromethane; (ii) coupling, 0.1 Mphosphoramidite and 0.45 M tetrazole in acetonitrile, 25 sec; (iii)capping, isobutyric anhydride/2,6-lutidine/THF 1/1/8 v/v/v as Cap A andstandard Cap B solution; (iv) sulfurization, 0.1M solution ofphenylacetyl disulfide (PADS) in acetonitrile:2,6-lutidine 1:1 mixture,5 mins. The oligonucleotide thio-phosphoramidates were cleaved from thesolid support and deprotected with concentrated aqueous ammonia. Thecompounds were analyzed and purified by HPLC. Ion exchange (IE) HPLC wasperformed using DIONEX DNAPac™ ion exchange column at pH 12 (10 mM NaOH)with a 1%/min linear gradient of 10 mM NaOH in 1.5 M NaCl and a flowrate of 1 ml/min. The products were desalted on Sephadex NAP-5 gelfiltration columns (Pharmacia) and lyophilized in vacuo. ³¹P NMRexperiments were performed in deuterium oxide to analyze the extent ofsulfurization analysis (31P NMR δ, ppm 58, 60 broad signals for Rp-,Sp-isomers).

Oligonucleotide thio-phosphoramidate 5′-GTTAGGGTTAG-3′ (SEQ ID NO.: 14)was synthesized the following way: An ABI Model 394 synthesizer was setup with 0.1M solutions of3′-tritylamino-2′,3′-dideoxy-N⁶-benzoyl-adenosine(N²-isobutyryl-guanosine, and thymidine)5′-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites. The reagent bottle ofstation #10 was filled with neat carbon disulfide and reagent bottle #15was filled with a solution of 15% S₈ in carbon disulfide containing 1%triethylamine. As the activator the commercially available 0.45 Msolution of tetrazole in acetonitrile was used. Cap A solution (station#11) was replaced by tetrahydrofuran/isobutyric anhydride/2,6-lutidine8/1/1 v/v/v solution. Cap B was also the commercially available reagent.A new function was created to deliver carbon disulfide from station #10to the column. The default sulfur synthesis cycle was modified thefollowing way: sulfurization time was set at 5 mins. The synthesiscolumn was filled with 1 μmole solid supportN²-isobutyryl-3′-(trityl)amino-2′,3′-dideoxyguanosine-5′-succinyl-loadedCPG (controlled pore glass). The sequence of the compound was programmedas GATTGGGATTG (5′→3′) (SEQ ID NO: 15). The trityl group was removed atend of the synthesis. The solid support was removed from the column andtreated with 1 ml concentrated aqueous ammonia at 55° C. for 6 hr in atightly closed glass vial. After filtration most of the ammonia wasevaporated and the remaining solution was desalted using Sephadex™ NAP-5gel filtration columns (Pharmacia) followed by lyophilization in vacuo.The product was analyzed and purified as described above.

EXAMPLE 4 Conjugation of Fluorescein to 3′-amino or 5′-aminoOligonucleotides

The reaction was conducted in 1.5 mL Eppendorff test tub, where ˜10-20OD of an oligonucleotide with free amino group was dissolved in 100 uLof 0.1 sodium-bicarbonate buffer, pH 8, and 50 uL of DMSO. Then ˜1 mg ofFITC (from Aldrich) was added to the solution, and the reaction mixturewas shaken by vortexing for 1-2 minutes. The reaction mixture was heatedto 55° C. for 30 minutes, and than left over night at room temperaturein darkness. The oligonucleotide product was precipitated in the sametest tube by the addition of 1.2 mL of EtOH, at −18° C. for 1 hour. Theprecipitate was separated from supernatant, and repricipitated twicewith EtOH from 100 uL of 1 M NaCl.

The obtained oligonucleotide conjugate was then analyzed and purified ifneeded by RP HPLC. Yields of the oligonucleotide conjugates with FITCwere 85-95%.

EXAMPLE 5 Conjugation of Ganciclovir to 3′-amino or 5′-aminoOligonucleotides

A 0.1 M solution of mono-O-dimethoxytritylganciclovir-O-CE-phosphoramidite in anhydrous acetonitrile was placed inan ABI Model 394 synthesizer using one of the extra stations forphosphoramidites. The synthesis and subsequent work up was carried outthe same way as described in Examples 2 and 3. Purity of the conjugatedoligonucleotide was assessed by ion exchange HPLC.

EXAMPLE 6 Preparation of Affinity Purified Extract Having TelomeraseActivity

Extracts used for screening telomerase inhibitors were routinelyprepared from 293 cells over-expressing the protein catalytic subunit oftelomerase (hTERT). These cells were found to have 2-5 fold moretelomerase activity than parental 293 cells. 200 ml of packed cells(harvested from about 100 liters of culture) were resuspended in anequal volume of hypotonic buffer (10 mM Hepes pH 7.9, 1 mM MgCl₂, 1 mMDTT, 20 mM KCl, 1 mM PMSF) and lysed using a dounce homogenizer. Theglycerol concentration was adjusted to 10% and NaCl was slowly added togive a final concentration of 0.3 M. The lysed cells were stirred for 30min and then pelleted at 100,000×g for 1 hr. Solid ammonium sulfate wasadded to the S100 supernatant to reach 42% saturation. The material wascentrifuged; the pellet was resuspended in one fifth of the originalvolume and dialyzed against Buffer ‘A’ containing 50 mM NaCl. Afterdialysis the extract was centrifuged for 30 min at 25,000×g. Prior toaffinity chromatography, Triton X-100™ (0.5%), KCl (0.3 M) and tRNA (50μg/ml) were added. Affinity oligonucleotide(5′-biotinTEG-biotinTEG-biotinTEG-GTA GAC CTG TTA CCA guu agg guu ag 3′[SEQ ID NO: 16]; lower case represents 2′ O-methyl ribonucleotides andupper case represents deoxynucleotides) was added to the extract (1 mmolper 10 ml of extract). After an incubation of 10 min at 30° C.,Neutravidin beads (Pierce; 250 μl of a 50% suspension) were added andthe mixture was rotated overnight at 4° C. The beads were pelleted andwashed three times with Buffer ‘B’ containing 0.3 M KCl, twice withBuffer ‘B’ containing 0.6 M KCl, and twice more with Buffer B containing0.3 M KCl. Telomerase was eluted in Buffer ‘B’ containing 0.3 M KCl,0.15% Triton X-100™ and a 2.5 molar excess of displacementoligonucleotide (5′-CTA ACC CTA ACT GGT AAC AGG TCT AC-3′ [SEQ ID NO:17] at 0.5 ml per 125 μl of packed Neutravidin beads) for 30 min. atroom temperature. A second elution was performed and pooled with thefirst. Purified extracts typically had specific activities of 10 fmolnucleotides incorporated/min/μl extract, or 200 nucleotides/min/mg totalprotein.

Buffer ‘A’ Buffer ‘B’ 20 mM Hepes pH 7.9 20 mM Hepes pH 7.9 1 mM MgCl2 1mM EDTA 1 mM DTT 1 mM DTT 1 mM EGTA 10% glycerol 10% glycerol 0.5 TritonX-100 ™

EXAMPLE 7 Telomerase Inhibition by Oligonucleotide Conjugates

Bio-Tel FlashPlate Assay

A suitable assay for detection and/or measurement of telomerase activityis based on a measurement of the addition of TTAGGG (SEQ ID NO. 8)telomeric repeats to a biotinylated telomerase substrate primer; areaction catalyzed by telomerase. The biotinylated products are capturedin streptavidin-coated microtiter plates. An oligonucleotide probecomplementary to 3.5 telomere repeats labeled with ³³P is used formeasuring telomerase products, as described below. Unbound probe isremoved by washing and the amount of probe annealing to the capturedtelomerase products is determined by scintillation counting.

The data provided in Tables 1 and 2 below were obtained by performingthe assay as described below:

1. Oligonucleotide conjugates were stored as concentrated stocks anddissolved in PBS.

2. For testing, the oligonucleotide conjugates were diluted to a 15×working stock in PBS and 2 μl was dispensed into two wells of a 96-wellmicrotiter dish (assayed in duplicate).

3. Telomerase extract was diluted to a specific activity of 0.04-0.09fmol dNTP incorporated/min./μl in Telomerase Dilution Buffer and 18 μladded to each sample well to preincubate with compound for 30 minutes atroom temperature.

4. The telomerase reaction was initiated by addition of 10 μl Master Mixto the wells containing telomerase extract and oligonucleotide compoundbeing tested. The plates were sealed and incubated at 37° C. for 90 min.

5. The reaction was stopped by the addition of 10 μl HCS.

6. 25 μl of the reaction mixture was transferred to a 96-wellstreptavidin-coated FlashPlate™ (NEN) and incubated for 2 hours at roomtemperature with mild agitation.

7. The wells were washed three times with 180 μl 2×SSC without anyincubation.

8. The amount of probe annealed to biotinylated telomerase products wasdetected in a scintillation counter.

Buffers:

Telomerase Dilution Buffer

50 mM Tris-acetate, pH 8.2

1 mM DTT

1 mM EGTA

1 mM MgCl₂

830 nM BSA

Master Mix (MM)

50 mM Tris-acetate, pH 8.2

1 mM DTT

1 mM EGTA

1 mM MgCl₂

150 mM K acetate

10 μM dATP

20 μM dGTP

120 μM dTTP

100 nM biotinylated primer (5′-biotin-AATCCGTCGAGCAGAGTT-3′) [SEQ IDNO.: 18]

5.4 nM labeled probe [5′-CCCTAACCCTAACCCTAACCC-(³³P) A₁₋₅₀-3′] [SEQ IDNO: 19]; specific activity approximately 10⁹ cpm/μg or higher

Hybridization Capture Solution (HCS)

12×SSC (1×=150 mM NaCl/30 mM Na₃Citrate)

40 mM EDTA

40 mM Tris-HCl, pH 7.0

Using the foregoing assay, various oligonucleotide conjugates weretested, and the data is presented in Tables 1 and 2. As can be seen, theaddition of the conjugated A group to the oligonucleotides typicallyenhanced the inhibitory activity of the oligonucleotide, sometimes byseveral orders of magnitude.

TABLE 1 Evaluation Of Oligonucleotide Conjugates As TelomeraseInhibitors (IC_(50 μM)) Oligonucleotide Conjugate NP NPS GGGTTAG(unconjugated)    0.6 0.008 GGGTTAG-L1-F    0.08 0.002 GGGTTAG-L2-F   0.06 0.003 GTTAGG (unconjugated)    0.47 0.12 GTTAGG-L1-F    0.20.016 GTTAGG-L2-F    1.5 0.015      TTAGGG (unconjugated)   11.0 1.2     TTAGGG-L1-F    0.034 0.003      TTAGGG-L2-F    2.0 0.009F-L2-TTAGGG    0.007 F-L2-TTAGGG-L1-F    0.005      TTAGGG-L2-Tr   11.5     TTAGGG-L1-Tr    0.4 0.04      TTAGGG-L1-X    0.14 0.09 A-L2-TTAGGG   1.0 0.06 A-L2-TTAGGG-L1-F    0.022 AGGG (unconjugated)  100,>1 0.087,0.05, 0.03 AGGG-L1-F   >1 0.018 GGGTTAG (unconjugated)    0.1, 1, 0.6*0.009, 0.005, 0.01 GGGTTAG-L1-F    0.1338, 0.0762, 0.196 0.002, 0.005,0.005 GGGTTAG-L2-F    0.059 0.003, 0.005 GGGTTAG-L1-Tr    0.436, 0.9330.029, 0.077 TGAGT (unconjugated)    0.399 0.036 TGAGT-L1-F    0.173,0.272 0.010, 0.029 GTAGGT    0.098, 0.162 0.140, 0.117 GTAGGT-L1-F   0.0156, 0.032, 0.015 0.099, 0.035, 0.033 Key: All conjugates shown inTable 1 were conjugated with a Type 1 or Type 2 linker to the 5′ or3′ sugar ring of the oligonucleotide sequence, represented by theplacement of the conjugated aromatic group to the left (5′) or right(3′) of the oligonucleotide sequence, respectively. NP indicates thatthe oligonucleotide has N3′→P5′ phosphoramidate internucleoside linkagesNPS indicates that the oligonucleotide has N3′→P5′ thio-phosphoramidateinternucleoside linkages L1: Type 1 linker (thiourea) L2: Type 2 linker(C5 or C6) Conjugated aromatic groups shown: F: fluorescein. Tr:N,N′-tetramethylrhodamine A: Acridine

TABLE 2 Evaluation Of Oligonucleotide Conjugates As TelomeraseInhibitors (IC₅₀ μM) Oligonucleotide Conjugate NP NPS GGGTTAG(unconjugated)  0.1, 1, 0.6, 0.081 0.009, 0.005, 0.010 GGGTTA-ORS1-G* 0.477, 0.626 0.026, 0.031 GGGTTA-ORS2-G*  0.005, 0.010 TTAGGG(unconjugated)  0.039, 0.079, >1 1.17, 1-55 TTAGG-ORS1-G* >1, 0.6910.305, 0.4 TTAGGG-ORS1-G* >1 0.0858, 0.108 TTAGG-ORS2-G*  0.004, 0.005,0.310, 0.115, 0.102, 0.24  0.721, >1, 5.15 TAA-ORS2-G*G*G*  0.004, 0.0090.102, 0.088 Key: All conjugates shown in Table 2 were conjugated with aType 3 linker (ORS1 or ORS2) to the 3′ sugar ring of the oligonucleotidesequence, represented by the placement of the conjugated aromatic groupto the right (3′) of the oligonucleotide sequence. NP indicates that theoligonucleotide has N3′→P5′ phosphoramidate internucleoside linkages.NPS indicates that the oligonucleotide has N3′→P5′ thio-phosphoramidateinternucleoside linkages. G* is the conjugated aromatic group guanine.ORS1 and ORS2 are Open Ring Sugar Linkers 1 and 2, respectively.TAA-ORS2-G*G*G* is a conjugated oligonucleotide in which a first guaninenucleobase is conjugated to the 3′ sugar of the oligoucleotide sequenceTAA through an ORS2 linker, and two additional guanine nucleobases areconjugated sequentially to the first guanine nucleobase through ORS2open ring sugar linkers.

EXAMPLE 8 Anti-Tumor Activity of Oligonucleotide Conjugates

Cell Assays

a. Inhibition of Telomerase Activity in Cells and Inhibition of TumorCell Growth

The following is a description of a general cell assay method that maybe used to determine the effect of the test conjugates on tumor cellgrowth. Colonies of human breast epithelial cells (spontaneouslyimmortalized) are prepared using standard methods and materials.Colonies are prepared by seeding 15-centimeter dishes with about 10⁶cells in each dish. The dishes are incubated to allow the cell coloniesto grow to about 80% confluence, at which time each of the colonies aredivided into two groups. One group was exposed to a subacute dose ofthio-phosphoramidate polynucleotide conjugate of experiment 9 at apredetermined concentration (e.g., between about 100 nM and about 20 μM)for a period of about 4-8 hours after plating following the split. Thesecond group of cells are similarly exposed to an unconjugated controloligonucleotide.

Each group of cells is then allowed to continue to divide, and thegroups are split evenly again (near confluence). The same number ofcellsare e seeded for continued growth. The test thio-phosphoramidateoligonucleotide conjugate or control oligonucleotide is added everyfourth day to the samples at the same concentration delivered initially.In certain experiments, the cells may also be treated with anoligonucleotide uptake enhancer, such as FuGENE6™ (Roche). Reduction oftelomerase activity associated with the oligonucleotide treatment isdetermined by TRAP assay.

In addition, telomere lengths in the treated cells may be determined bydigesting the DNA of the cell samples using restriction enzymes specificfor sequences other than the repetitive T₂AG₃ sequence of humantelomeres (TRF analysis). The digested DNA is separated by size usingstandard techniques of gel electrophoresis to determine the lengths ofthe telomeric repeats, which appear, after probing with a telomere DNAprobe, on the gel as a smear of high-molecular weight DNA (approximately2 Kb-15 Kb).

These approaches were used to determine the effect of treatment with theconjugate TTAGGG-L1-F on HME50 and Caki-1 cells. IC₅₀ values in therange of ca. 5 μmole (in the range of 1-20 μmole) were obtained in theabsence of an uptake enhancer. When cells were incubated with thisconjugate in the presence of FuGENE6 they underwent cell crisis andapoptosis after approximately 15 days of treatment. Growth of controlcells was not affected.

b. Specificity

The short oligonucleotide conjugates of the invention are screened foractivity (IC₅₀) against telomerase and other enzymes known to have RNAcomponents by performing hybridization tests or enzyme inhibition assaysusing standard techniques. Oligonucleotides having lower IC₅₀ values fortelomerase as compared to the IC₅₀ values toward the other enzymes beingscreened are said to possess specificity for telomerase.

c. Cytotoxicity

The cell death (XTT) assay for cytotoxicity are performed usingHME50-5E, Caki-1, A431, ACHN, and A549 cell types. The cell lines usedin the assay are exposed to one of the short oligonucleotide conjugatesfor 72 hours at concentrations ranging from about 1 μM to about 100 μMin the presence and absence of lipids. During this period, the opticaldensity (OD) of the samples is determined for light at 540 nanometers(nm). The IC₅₀ values obtained for the various cell types are generallyless than 1 μM. Thus, no significant cytotoxic effects are expected tobe observed at concentrations less than about 100 μM. It will beappreciated that other tumor cells lines such as the ovarian tumor celllines OVCAR-5 and SK-OV-3 can be used to determine cytotoxicity inaddition to control cell lines such as normal human BJ cells. Otherassays for cytotoxicity such as the MTT assay (see Berridge et al.,Biochemica 4:14-19, 1996) and the alamarBlue™ assay (U.S. Pat. No.5,501,959) can be used as well.

Preferably, to observe any telomerase inhibiting effects theoligonucleotide conjugates should be administered at a concentrationbelow the level of cytotoxicity. Nevertheless, since the effectivenessof many cancer chemotherapeutics derives from their cytotoxic effects,it is within the scope of the present invention that the oligonucleotideconjugates of the present invention be administered at any dose forwhich chemotherapeutic effects are observed.

In Vivo Animal Studies

A human tumor xenograft model in which OVCAR-5 tumor cells are graftedinto nude mice can be constructed using standard techniques andmaterials. The mice are divided into two groups. One group is treatedintraperitoneally with a short oligonucleotide conjugates of theinvention. The other group is treated with a control comprising amixture of phosphate buffer solution (PBS) and an oligonucleotidecomplementary with telomerase RNA but has at least a one base mismatchwith the sequence of telomerase RNA. The average tumor mass for mice ineach group is determined periodically following the xenograft usingstandard methods and materials.

In the group treated with oligonucleotide conjugates of the invention,the average tumor mass is expected to increase following the initialtreatment for a period of time, after which time the tumor mass isexpected to stabilize and then begin to decline. Tumor masses in thecontrol group are expected to increase throughout the study. Thus, theoligonucleotide conjugates of the invention are expected to lessendramatically the rate of tumor growth and ultimately induce reduction intumor size and elimination of the tumor.

Thus, the present invention provides novel oligonucleotide conjugatesand methods for inhibiting telomerase activity and treating diseasestates in which telomerase activity has deleterious effects, especiallycancer. The oligonucleotide conjugates of the invention provide a highlyselective and effective treatment for malignant cells that requiretelomerase activity to remain immortal; yet, without affectingnon-malignant cells.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A method of inhibiting a telomerase enzyme, comprising: contacting acancer cell that expresses telomerase with a compound comprising anoligonucleotide conjugated to a nucleobase, having the structure:(A—L—)_(n)—O wherein A is a single nucleobase, n=1 or 2, and L is aflexible linker group, such that A—L does not represent a nucleoside ornucleotide, and L does not include a 5- or 6-membered closed ring sugargroup; O is an oligonucleotide consisting of 4 to 15 nucleoside subunitsjoined by N3′→P5′ phosphoramidate or N3′→P5′ thiophosphoramidateintersubunit linkages and comprising a sequence of 4 or more consecutivenucleosides exactly complementary to the template region of humantelomerase RNA; and wherein the compound inhibits telomerase activity.2. The method of claim 1, wherein A is conjugated via linker L to the 3′nucleoside of oligonucleotide O, and linker L has the structure:

wherein: X=N or O; Y=O or S; Z=O or S; W=N, O, S or lower alkyl; V loweralkyl; Q=O, S or NR′″, wherein R′″ is H, lower alkyl or lower acyl; R′and R″ are independently ═H, OH, alkyl or alkylamine; and wherein X isconjugated to the sugar ring of the 3′ nucleo side of oligonucleotide O.3. The method of claim 2, wherein the linker L is selected from thegroup consisting of:

wherein Y=O or S.
 4. The method of claim 3, wherein A is guanine.
 5. Themethod of claim 1, wherein n=1 and A—L is selected from the groupconsisting of:


6. A method of inhibiting a telomerase enzyme, comprising: contacting acancer cell that expresses telomerase with a compound comprising anoligonucleotide conjugated to a nucleobase, having the structure:(A—L—)_(n)—O wherein A is a single nucleobase, n=1 or 2, and L is aflexible linker group, such that A—L does not represent a nucleoside ornucleotide, and L does not include a 5- or 6-membered closed ring sugargroup; O is an oligonucleotide composed of nucleoside subunits joined byN3′→P5′ phosphoramidate or N3′→P5′ thiophosphoramidate intersubunitlinkages and comprising a sequence selected from the group consistingof: TAGGGTTAGACAA (SEQ ID: 3); GTTAGGGTTAG (SEQ ID: 4); AGGGTTAG (SEQID: 5); GGGTTAG (SEQ ID: 6); GTTAGG (SEQ ID: 7); TTAGGG (SEQ ID: 8); TTA(SEQ ID: 9); AGGG (SEQ ID: 10); GGGTTA (SEQ ID: 11); TGAGTG (SEQ ID:12); and GTAGGT (SEQ ID: 13) and wherein the compound inhibitstelomerase activity.
 7. The method of claim 1, wherein the compound isformulated with a penetration enhancing agent.
 8. The method of claim 1,wherein n is 1, and the nucleobase A is conjugated to the 3′ nucleosideof the oligonucleotide O via linker L.
 9. The method of claim 6, whereinL is a linker of structure:

wherein: X=N or O; Y=O or S; Z=O or S; W=N, O, S or lower alkyl; V=loweralkyl; Q=O, S or NR′″, wherein R′″ is H, lower alkyl or lower acyl; andR′ and R″ are independently ═H, OH, alkyl or alkylamine.
 10. The methodof claim 1, wherein the oligonucleotide O is 4-8 nucleosides in lengthand is exactly complementary to the template region of human telomeraseRNA.
 11. A method of inhibiting a telomerase enzyme, comprising:contacting a cancer cell that expresses telomerase with a compoundcomprising an oligonucleotide conjugated to a fluorophore, having thestructure:(A—L—)_(n)—O wherein A is a fluorophore, L is a linker group, O is anoligonucleotide consisting of 4 to 15 nucleoside subunits joined byN3′→P5′ phosphoramidate or N3′→P5′ thiophosphoramidate intersubunitlinkages and comprising a sequence of 4 or more consecutive nucleo sidesexactly complementary to the template region of human telomerase RNA; nis an integer between 1 and m+1, where m is the total number ofnucleosides in O; and wherein the compound inhibits telomerase activity.12. The method of claim 11, wherein A is selected from the group

consisting of: wherein X, X₁ and X₂ are independently selected from thegroup consisting of O, S, and N; X₃ is hydrogen, halogen, or alkyl; X₄is C, N, O, or S; X₁R₁ and X₂R₂ are independently selected from thegroup consisting of hydroxyl (OH) or a salt thereof, thiol (SH) or asalt thereof, methoxy, methylthio, ethoxy, ethylthio, propoxy, andpropylthio; and R₃, R₄ and R₅ are independently selected from the groupconsisting of H, hydroxyl, halogen, alkyl, aryl, carboxyl, and X-alkyl.13. The method of claim 12, wherein L is a linker having the structure:

wherein Z₁, Z₂, and Z₃ are independently selected from O, S, and NR4,where R4 is H or lower alkyl, and Z₁ and Z₃ can be direct bonds.
 14. Themethod of claim 12, wherein L is a linker comprising the structure:—(CH₂)₆(OPO₂ ⁻)——(CH₂)₄CH(CH₂OH)(CH₂OPO₂ ⁻)— or—(CH₂)₄CH(CH₂OH)(CH₂OP(S)O₂ ⁻)—.
 15. The method of claim 13, wherein Lis a thiourea linkage.
 16. The method of claim 12, wherein A is selectedfrom the group consisting of fluorescein, N,N′-tetramethylrhodamine, andacridine.
 17. A method of inhibiting a telomerase enzyme, comprising:contacting a cancer cell that expresses telomerase with a compoundcomprising an oligonucleotide conjugated to a fluorophore, having thestructure:(A—L—)_(n)—O wherein A is a fluorophore, n is an integer between 1 andm+1, where m is the total number of nucleosides in O; L is a linkergroup, O is an oligonucleotide composed of nucleoside subunits joined byN3′→P5′ phosphoramidate or N3′→P5′ thiophosphoramidate intersubunitlinkages and comprising a sequence selected from the group consistingof: TAGGGTTAGACAA (SEQ ID: 3); GTTAGGGTTAG (SEQ ID: 4); AGGGTTAG (SEQID: 5); GGGTTAG (SEQ ID: 6); GTTAGG (SEQ ID: 7); TTAGGG (SEQ ID: 8); TTA(SEQ ID: 9); AGGG (SEQ ID: 10); GGGTTA (SEQ ID: 11); TGAGTG (SEQ ID:12); and GTAGGT (SEQ ID: 13) and wherein the compound inhibitstelomerase activity.
 18. The method of claim 11, wherein theoligonucleotide O is 4-8 nucleosides in length and is exactlycomplementary to the template region of human telomerase RNA.